A central problem for the development of HIV-1 vaccines has been to identify immunogens capable of raising high titer, long lasting, neutralizing antibody to the envelope glycoproteins (Envs) of primary isolates. The HIV-1 Env has proven to be a weak immunogen, raising low titer antibodies that are slow to undergo affinity maturation. In this proposal, DNA immunogens will be used to test soluble forms of CD4 (sCD4) fused to Env in order to generate neutralizing antibodies to protected regions. In HIV/cell interactions, Env attaches to CD4 on the cell surface. This interaction leads to changes in Env, which expose cryptic regions important for Env binding to co-receptors and subsequent fusion and entry into human cells. DNA immunogens of sCD4/Env will allow for the stable exposure of Env regions for generation of neutralizing antibodies (Ab). In addition, in order to enhance the neutralizing antibody response, DNA immunogens will be tested containing sCD4/Env fused to C3d. HIV-1 Env has been a target for developing an effective vaccine. Our laboratory has successfully used the C3d component of complement as a molecular adjuvant for viral glycoproteins. In normal immune responses, the attachment of Gd to an antigen enhances both the initiation and the maturation of antigen-specific antibody. To test a potential role of sCD4 and C3d for Env, DNA constructs will be produced encoding Env fused to sCD4 (sCD4/Env) and sCD4/Env/C3d and used for immunizations. The study will be executed according to three specific aims. (1) Vaccine plasmids encoding a secreted monomeric (gpl20) form of primary Envs and these same forms of Env fused at the amino terminus with one of two forms for sCD4 (2 domain or 4 domain) and/or at the carboxyl terminus to three tandem copies of C3d will be constructed. The levels of expression and secretion of the various plasmid constructs will be determined. (2) The Env constructs will be compared for immunogenicity in rabbits, using DNA immunization. Raised antibody will be titered for the level of Env-specific IgG and for neutralizing activity for a panel of primary HIV-1 isolates. (3) Codon-optimized Env genes will be used in these same constructs and efficient expression and immunogenicity will be determined. Our goal is to identify the most favorable sCD4/Env/C3d fusion for raising high titer neutralizing antibody. The hypothesis throughout the study is that the sCD4 fusion will increase the neutralizing Ab response and the C3d fusion will enhance the immunogenicity of Env both by increasing the efficiency of the initiation of anti-Env Ab response and by increasing the ability of anti-Env Ab to undergo affinity maturation.